Regulated actin polymerization is critical for driving cellular behaviors, such as locomotion and phagocytosis that play a critical role in embryonic development, immune system function and tissue repair. These cellular behaviors are also important factors in the pathogenesis of cancer and cardiovascular disease. The long-term goal of this project is to understand the function of key molecules that control actin polymerization in cells and to determine how they are regulated. The specific goal is to dissect the mechanism of function and regulation of the human Arp2/3 complex. This protein complex plays a central role in nucleating actin filaments and organizing them into Y-branched arrays in lamellipodia and other cellular structures. The Arp2/3 complex is activated by a class of proteins called nucleation promoting factors (NPFs). These, in turn, are regulated by signal transduction molecules and other proteins that coordinate the spatial and temporal distribution of actin nucleation in cells. However, we do not understand key aspects of the mechanism by which the Arp2/3 complex nucleates and organizes actin filaments, or how Arp2/3 complex activity is regulated by nucleation promoting factors and their interacting partners. We will take a biochemical and cell biological approach to address these important outstanding issues. The Specific Aims are to: (1) map functionally critical surface regions on the Arp2/3 complex and define its activation mechanisms, (2) elucidate the molecular mechanism of Arp2/3 complex regulation by the Wiskott-Aldrich Syndrome protein (WASP), and (3) define the function and regulation of KIAA1971, a novel nucleation promoting factor. In the long term, these experiments will help elucidate how actin nucleation is controlled in cells, and how the proteins that control nucleation act with factors that modulate other aspects of actin dynamics to drive processes such as membrane protrusion during cell locomotion.